Myelin is a fundamental component of mature neural networks that is affected in a large number of pathological conditions of the central nervous system (CNS). Critical for advancing knowledge about these conditions would be a better in vivo understanding of how oligodendrocytes and their respective myelin sheaths develop, are maintained throughout life and respond to injury. Growing evidence suggests neuronal activity and proper astrocyte function may play crucial roles in these processes. We have developed a new technique that allows high resolution label-free in vivo imaging of myelinated axons. This technique takes advantage of the high refractive index of lipid rich multilayered myelin and is based on spectral confocal reflectance (SCoRe) microscopy. Using SCoRe imaging I have obtained for the first time long-term images of the dynamics of cortical myelin on the cellular scale in a living animal. Preliminary data shows this technique as well as fluorescence imaging of oligodendrocytes, axons and astrocytes are a powerful set of tools that in combination provide a wealth of information about fine structural dynamics of these structures in vivo. I demonstrate the feasibility to track long- term changes in internode length in addition to documenting the temporal dynamics of demyelination after single oligodendrocyte and astrocyte ablation. I propose to use these powerful techniques to address three fundamental questions concerning the in vivo plasticity and regeneration of myelin and oligodendrocytes. First, I will determine the long term plasticity of myelin and oligodendrocytes in the mouse cortex. Next I will determine the effects of neuronal activity on myelin formation, plasticity and oligodendrocyte regeneration. Finally I will use single cell ablation techniques to determine if astrocytes are required for or alter the temporal dynamics of oligodendrocyte remyelination in vivo. Together these experiments will describe for the first time the longitudinal dynamics and regeneration capabilities of oligodendrocytes and myelin on the cellular scale. Furthermore these experiments will reveal how changes in axons and astrocytes influence these dynamics over weeks to months in the living brain.